Project Summary Title: Dual mechanism thiopeptide analogs for TB drug development Tuberculosis (TB) is a global pandemic: 10.4 million new cases and 1.8 million deaths from TB in 2015. The TB health crisis is compounded by the rise in multidrug resistant and extensively drug resistant (MDR and XDR) strains of Mycobacterium tuberculosis (Mtb), the causative agent of TB. There is an urgent need to develop new drugs with novel mechanisms of action and improved efficacy. Thiopeptides are natural products discovered to have anti-Mtb activity over 60 years ago. Thiopeptides have potent activity and they act by inhibiting bacterial protein synthesis through a novel mechanism that avoids cross-resistance with other antibiotics. However, because thiopeptides manifest extremely poor aqueous solubility, their development as anti-TB drugs was abandoned long ago. Recent data shows thiopeptides to also be able to induce autophagy in macrophages. Because autophagy can serve as a host defense to eliminate intracellular pathogens, such as Mtb, this thiopeptide activity could enhance host antimicrobial mechanisms (i.e. serve as a host directed therapeutic) to supplement the direct anti-Mtb effects of thiopeptides. Such a dual mode of action of a single compound (bacterial and host effects) has the exciting potential to provide two therapeutic effects with one drug. In this R21, we will revisit the therapeutic potential of thiopeptides. In Aim 1, we will use our innovative chemoenzymatic approach to thiopeptide synthesis to produce non-natural thiopeptide analogs with improved aqueous solubility. Analogs will be tested for activity against Mtb in vitro and for autophagy inducing activity on macrophages. Quantitative structure-activity relationship modeling will inform subsequent rounds of synthesis and improvement. In Aim 2, we will test the most tractable compounds for efficacy on Mtb infected macrophages and assess the contribution of the dual mechanisms of action to thiopeptide control of intracellular Mtb. The outcome of the proposed Aims, will be the identification of thiopeptide analogs with improved solubility, anti-Mtb activity and host directed activity that can serve as lead compounds for future preclinical testing in animal models. This study will advance ongoing efforts to develop new TB drugs with novel mechanisms of action, including host directed effects that could synergize with standard antibacterials. Because thiopeptides are also effective on several Gram-positive pathogens, our results may have additional implications for a wider array of infectious diseases.